Method of tissue repair using a multi-layered matrix

ABSTRACT

A multi-layered matrix, a method of tissue repair using the same, and multi-layered implant prepared thereof are provided. The multi-layered matrix comprises a first element and a second element connected thereto, and the second element comprises a hollow cavity. The first and the second elements are composed of a composite material comprising a bioabsorbable porous material.

This application is a Divisional of application Ser. No. 11/413,020,filed on Apr. 28, 2006now abandoned, which claims priority under 35U.S.C. §119(a) of Patent Application No. 94147253 filed in Taiwan R.O.C.on Dec. 29, 2005, the entire contents of which are hereby incorporatedby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to tissue repair, and in particularmulti-layered matrix combined with cell blocks for tissue repair.

2. Description of the Related Art

Articular cartilage formed on the articular extremities, or surface ofbones is a multi-functional tissue and due to elastic property, canbreak the force of concussions, lubricate the surface of bones with lowfriction coefficient, and enable perfect ease and freedom of movementbetween the bones. Cartilage cells, chondrocytes, are about 2% by weightof the articular cartilage and covered with plenty of extracellularmatrices. The major difference of articular cartilage to other tissuesis that it does not contain blood-vessels, lymphatic vessels, or nerves.Cartilage metabolism is relatively slow in comparison with othertissues; hence, it is much more difficult for defects in cartilage toheal spontaneously. Patients with articular cartilage defects may notfeel pain since no nerve is distributed in the articular cartilage.Chondrocytes covered by cell matrices are well-differentiated cells andhave low division ability. In addition, mesenchymal stem cells will notbe evoked and migrated to the injured area since cartilage lacks bloodvessels and lymphatic vessels.

Articular cartilage defects can be classified into partial thicknessdefect and full thickness defect according to their severity.Partial-thickness defect is a lesion or erosion on the cartilage tissueof the articular surface that does not reach the subchondral bonewhereas full-thickness defect penetrates the subchondral bone. With theadvances in surgery and arthroscopy, partial thickness defects may betreated or its symptoms may be relieved by surgery or arthroscopicmethods such as abrasion arthroplasty, debridement and lavage, hightibial osteotomy, however, these surgeries cannot treat severe damagesuch as full thickness defects. As a result, patients are faced with theonly choice of undergoing both joint excision and replacement with anartificial joint to relieve the pain and regain joint function. In theUnited States, it is estimated that over 150,000 knee replacementoperations caused by full thickness defects annually and the number ofsuch operations is increasing year by year. Artificial joints areexpensive as is replacement operation. In addition, artificial jointsmade of metal only have a ten- to twenty-year life-span. For youngpatients, a second replacement operation is inevitable, however, olderpatients may not be able to receive a second replacement operation andbecome disabled at the rest of their life. Development of a newtreatment for full thickness defects of cartilage is therefore veryimportant.

Methods available to treat cartilage full thickness defects includemicrofracturing and drilling. This technology is a marrow stimulatingarthroscopic procedure to penetrate the subchondral bone to inducefibrin clot formation and the migration of primitive stem cells from thebone marrow into the defective cartilage location. More particularly,the base of the defective area is shaved or scraped to induce bleeding.An arthroscopic awl or pick is then used to make small holes ormicrofractures in the subchondral bone plate. The end of the awl ismanually struck with a mallet to form the holes while care is made notto penetrate too deeply and damage the subchondral plate. The holespenetrate a vascularisation zone and stimulate the formation of a fibrinclot containing pluripotential stem cells. The clot fills the defect andmatures into fibrocartilage. Microfracturing the subchondral bone platecan be a successful procedure for producing fibrocartilaginous tissueand repairing defective articular cartilage, however, it still has somedisadvantages. For example, the microfractures or holes are manuallycreated. If the holes are not deep enough, then the formation of thefibrin clot may not occur. On the other hand, if the holes are too deep,the subchondral bone plate can be damaged and lead to unwantedconsequences and complications. In addition, the fibrocartilageformation may fill the defects, but the cartilage function cannot betotally restored. Another technology is Mosaic Plasty proceduredeveloped by a Hungarian surgeon in 1995. This technique involves usinga series of dowel cutting instruments to harvest a plug of articularcartilage and subchondral bone from a donor site, which can then beimplanted into a core made into the defect site. By repeating thisprocess, transferring a series of plugs, and by placing them in closeproximity to one another, in mosaic-like fashion, a new grafted hyalinecartilage surface can be established. The result is a hyaline-likesurface interposed with a fibrocartilage healing response between eachgraft. The advantages of this technique include the grafts are thepatient's own tissue and allograft or xenograft rejection can beprevented. In addition, the grafts are biphasic joint containingcartilage and bone and can be implanted to the articular surface toprovide excellent support while the surrounding bone tissue grows intothe bone portion of the grafts. This procedure, however, is technicallydifficult. In addition, the grafts are obtained from the unstressed areaof the patient, which is limited to a restrained area. The grafting mayalso destroy the integrity of the joint.

Recently, a new approach for restoration of articular cartilage defectsby ex vivo multiplied autologous cartilage has been developed.Chondrocytes from healthy articular cartilage are harvested and theextracellular matrices are digested by enzymes. Chondrocytes aremultiplied outside the body for 11 to 21 days to be more than ten timesthe original number. The cell concentration is adjusted to 2.6×10⁶-5×10⁶cells/ml, and the cells are then injected into the defect site coveredwith a layer of periosteum by suturing prior to the injection. Thistechnique is under clinical trial, however, and faces a problem in thatchondrocytes are dedifferentiated during the ex vivo multiplicationstep. The originally rounded chondrocytes become spindle-shapedfibrocartilages and the biochemical properties of the cells are alsoaltered. In addition, the steps of obtaining and suturing autologousperiosteum cannot be performed under endoscope. Moreover, it requires atleast two surgical procedures (i.e., one to harvest the cells and one toreimplant them); it is relatively expensive; and there are limits in thesize of lesion, and the number of lesions, that can be treated.

Other techniques combine materials and cells to repair full thicknessdefects in cartilage or bone. Biomedical materials are selected based onthe physical and mechanical properties of cartilage or bone. Forcartilage, naturally occurring or synthetic bioabsorbable polymericmaterials are selected, such as collagen, gelatin, alginate, poly(glycolide), poly (lactide) (PLLA), poly (glycolide co-lactide) (PLGA).For bone, biomedical ceramic materials are selected, such ashydroxyapatite, tricalcium phosphate, calcium carbonate, or calciumsulfate. The combination of bioabsorbable polymeric materials andbiomedical ceramic materials to mimic bones has also been proposed. Asfor the structure of cartilage, porous structure is prepared tointroduce surrounding tissues thereto or as a scaffold for the implantedcells. In addition, the combination of chondrocytes and gel to from ahydrogel with cells has been proposed. The hydrogel with cells can beattached on the bone-layer material to from a biphasic structure of boneand cartilage. The bone-layer material is also porous to introducesurrounding bone tissue thereto since bone tissue has a strongerregeneration ability than cartilage. As for the combination of materialsand cells, small amount of autologous cartilage tissue is harvested,digested with enzymes to remove extracellular matrices and releasechondrocytes, and the chondrocytes are implanted into a porous scaffoldfor multiplication. An appropriate amount of multiplied chondrocytes arethen implanted to the defect site. In general, this technique is usedfor simple evenly-distributed tissue, not for multi-layered tissue. Whentwo different cells are implanted in a porous matrix, cells may flow andmix since the cell size is smaller than the pore size of the matrix. Therecent technique for multi-layer cultivation involves ex vivomultiplication of cartilage and bone tissues separately, implantation ofthe multiplied cartilage and bone tissues to two different porousmatrices respectively, combination of the two matrices containingcartilage and bone tissues, and fusion of the borders of the twomatrices by refusion cultivation to form a biphasic matrix. Thistechnique is, however, time-consuming and also not clinically appliedyet.

It therefore would be advantageous to provide a more effective method oftissue repair using a multi-layered matrix.

BRIEF SUMMARY OF THE INVENTION

A detailed description is given in the following embodiments withreference to the accompanying drawings.

Accordingly, the invention provides a multi-layered matrix and a methodof tissue repair using the same to solve the drawbacks of theconventional articular cartilage restoration. The multi-layered matrixwas designed to have differential structures to grow different cells indifferent portions of the matrix by the structure of the matrix and thevolume differences of tissues. The matrix containing multi-layeredtissues can be implanted into the defect site of a subject to repair thedefect site with the tissues.

An embodiment of the invention provides a multi-layered matrix. Thematrix comprises a first element and a second element connected thereto,and the second element comprises a hollow cavity. The first and thesecond elements are composed of a composite material comprising abioabsorbable porous material. The porous material of the first elementis for the growth of the surrounding cells of an implanted site in aliving subject, and the hollow cavity of the second element is seededwith a cell block prior to the implantation of the living subject.

Also provided is a method of tissue repair using the multi-layeredmatrix. The method comprises providing the multi-layered matrix, seedinga cell block into the hollow cavity of the multi-layered matrix, andimplanting the multi-layered matrix containing the cell block into adefect site of a living subject.

Further provided is a multi-layered implant prepared the method as abovedescribed.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

The present invention can be more fully understood by reading thesubsequent detailed description and examples with references made to theaccompanying drawings, wherein:

FIGS. 1A and 1B are photographs of the multi-layered matrix of anembodiment of the invention. FIG. 1A is a lateral view and FIG. 1B is across-section view.

FIGS. 2A˜2C are electron microscopic photographs of the multi-layeredmatrix of the embodiment of the invention. FIG. 2A shows the porousstructure surrounding the hollow cavity, FIG. 2B shows the porousstructure beneath the hollow cavity, and FIG. 2C shows the interfacebetween the porous structures surrounding and beneath the hollow cavity.

FIGS. 3A and 3B are microscopic photographs of articular cartilage. FIG.3A shows the minced cartilage, and FIG. 3B shows the cartilage beingdigested by enzymes for 4 hours after minced.

FIGS. 4A and 4B are photographs showing the multi-layered matrix of theembodiment of the invention being seeded with the digested cartilage,implanted into the back of a nude mouse, and retrieved from the mouseafter one month. FIG. 4A is a lateral view, and FIG. 4B is across-section view.

FIGS. 5A˜5E are histological photographs showing the multi-layeredmatrix of the embodiment of the invention being seeded with the digestedcartilage, implanted into the back of a nude mouse, and retrieved fromthe mouse after 1 month. FIGS. 5A and 5B shows HE staining, FIG. 5Cshows safranin O staining, FIG. 5D shows collagen type II staining, andFIG. 5E shows collagen type I staining.

FIGS. 6A and 6B are photographs showing the surgical procedure forimplantation of a biphasic implant containing chondrocytes into the kneejoint of a Lee-Sung strain miniature pig. FIG. 6A shows a defect formedon the knee joint of the Lee-Sung strain miniature pig, FIG. 6B shows abiphasic implant containing chondrocytes implanted into the defect site.

FIG. 7 is a photograph showing two biphasic implants obtained from theLee-Sung strain miniature pig after a half-year implantation. The leftside is the control group and the right side is the experimental group.

FIGS. 8A and 8B are X-ray results showing the joint after theimplantation of the biphasic implant of the embodiment of the inventionfor half year. FIG. 8A is the control group, and FIG. 8B is theexperimental group.

DETAILED DESCRIPTION OF THE INVENTION

The following description is of the best-contemplated mode of carryingout the invention. This description is made for the purpose ofillustrating the general principles of the invention and should not betaken in a limiting sense. The scope of the invention is best determinedby reference to the appended claims.

A multi-layered matrix, a method of tissue repair using the same, amulti-layered implant prepared thereof are provided.

The multi-layered matrix of the embodiment of the invention is abiphasic matrix which can be implanted into a defect of cartilage andbone tissues. A hollow cavity is on the upper side of the matrix fortissue block containing cells. The size differences of the tissue andthe porous structure of the matrix facilitate controlled distribution tocentralize the chondrocytes in the upper cavity of the biphasic matrix.The biphasic matrix is then implanted into a defect site of a livingsubject to reconstruct tissue therein.

The inventors have developed a method and carrier for culturingmulti-layered tissue in vitro. The method comprises providing a porousmulti-layered carrier having a hollow cavity, placing tissue blockswithin the hollow cavity of the porous multi-layered carrier, seedingcells into the carrier, and incubating the tissue blocks and cellswithin the carrier. With the structure of the carrier and the volumedifferences between tissue blocks and cells, the tissue blocks and cellscan be grown into a two-layered cartilage tissue in vitro formulti-layered tissue repair. The inventors further modified the methodand carrier as above described and found that the multi-layered porousmatrix containing cells or tissue blocks can be directly implanted intothe defect site of a living subject without the ex vivo incubation step.The multi-layered porous matrix containing tissue blocks or cells werethen subjected to Mosaic Plasty procedure. A fillister having similarsize to the biphasic matrix was created on the defect site of a livingsubject. An autologous cartilage tissue block was collected from theunstressed area of the articular cartilage. For enhancing the fillingarea, the cartilage tissue was minced into small pieces and digested byenzymes to release chondrocytes. The partially digested tissue blockswere then placed into the hollow cavity of the biphasic matrix. Tissueblocks were centralized in the upper hollow cavity since the pore sizeof the porous structure surrounding the hollow cavity is smaller thanthe tissue blocks. The biphasic implant containing the tissue blocks wasthen implanted into the fillister on the defect of the articularcartilage to regenerate tissue and repair the articular cartilagedefect. Similarly, autologous chondrocytes can be multiplied in vitro,the cell blocks or cells combined with gel or other biomaterials can beplaced into the hollow cavity of the biphasic matrix, and the biphasicmatrix containing the cells can be implanted into a fillister created onthe defect site to repair large defect area.

With the embodiment of the method of tissue repair of the invention,only a small amount of cartilage tissue rather than bone tissue arecollected for the implant, avoiding destruction of the integrity andmechanical property of the joint. In addition, the collected tissueblocks are minced and digested with enzyme, enlarging the repair areaand enhancing cell propagation and fusion effects. Moreover, this methodis an one-step operation which can be manipulated under endoscope toreduce pain and hospitalization time.

Accordingly, an embodiment of the invention provides a multi-layeredmatrix. The matrix comprises a first element and a second elementconnected thereto, and the second element comprises a hollow cavity. Thefirst and the second elements are composed of a composite materialcomprising a bioabsorbable porous material. It is provided that theporous material of the first element is for the growth of thesurrounding cells of an implanted site in a living subject, and thehollow cavity of the second element is seeded with a cell block prior tothe implantation of the living subject to restore the implant site.

The composite material comprising the bioabsorbable porous materialincludes, but is not limited to, polylactic acid (PLA), polyglycolicacid (PGA), poly(glycolide co-lactide) (PLGA), polyanhydride,polycapralactone (PCL), polydiester, polyorthoester, collagen, gelatin,hyaluronic acid, chitosan, or polyethylene glycol (PEG), preferablypoly(glycolide co-lactide) (PLGA). The pore size of the porous matrixranges from 50 to 1000 μm. The composite material may further compriseother materials, including, but are not limited to, hydroxyapatite(HAP), tricalcium phosphate (TCP), tetracalcium phosphate (TTCP),dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrate(DCPD), octacalcium phosphate (OCP), or calcium pyrophosphate (CPP),preferably tricalcium phosphate (TCP).

The embodiment of the multi-layered matrix, when used for joint defectrepair, can be implanted into a joint of a living subject. The cellblock can be cartilage. The cartilage can be obtained from theimplantation subject or from other living organisms. In addition, thecartilage tissue can be incubated ex vivo. The size of the cell blocksrange from 100 to 2,000 μm.

Also provided is a method of tissue repair using the multi-layeredmatrix. The method comprises providing the multi-layered matrix, seedinga cell block into the hollow cavity of the multi-layered matrix, andimplanting the multi-layered matrix containing the cell block into adefect site of a living subject.

The cell block can be a minced tissue block, a tissue and cell aggregateprepared from a tissue block minced and digested with enzymes to releasepartial cells from the tissue block, or an in vitro multiplied cellblock combined with gel. The enzyme digestion time ranges from 5 min to24 hours. The enzymes include, but are not limited to, collagenase,hyaluronidase, trypsin, or proteinase. When the defect site is locatedon a joint, the cell block can be cartilage and the enzyme can becollagenase.

The cell block can be a cell aggregate from in vitro cultivation of acell. When the implant site is a joint defect, a chondrocyte can be invitro cultured to form the cell aggregate.

Moreover, the cell block can be a granular carrier attached with cellsif necessary.

It is to be noted, the size of the cell block is larger than the poresize of the porous material. For example, the size of the cell blockranges from 100 to 2,000 μm.

Further provided is a multi-layered implant prepared by the methoddescribed above.

Practical examples are described herein.

EXAMPLES Example 1 Preparation of Porous Matrix

The biodegradable polymer used herein was poly (glycolide co-lactide)(PLGA) prepared by ring-open polymerization with a molecular weight of580,000 determined by gel permeation chromatography. PLGA was mixed witha biomedical ceramic powder, tricalcium phosphate (TCP). Sodium chlorideparticle with diameter 250 μm was also added to produce apertures. Theorganic solvent for dissolving polymeric particles was acetone.

Two grams of PLGA was dissolved in 40 ml of acetone. Eight gram ofsodium chloride was then added to the solution with a ratio ofPLGA/NaCl:20/80% by weight. The mixture was poured into a Teflon moldwith a size of 10 cm×10 cm×0.5 cm and then placed in a laminar flow toevaporate the organic solvent. PLGA film containing NaCl particles andhaving a thickness of 0.8 mm was obtained and trimmed into round sliceswith a diameter of 8.5 mm and strips with a size of 27 mm×3 mm.

PLGA blocks were evenly dissolved in acetone. Tricalcium phosphate (TCP)was then added to the solution with a ratio of PLGA/TCP:50/50% byweight. The sticky mixture was poured into a Teflon mold and then placedin a laminar flow to evaporate the organic solvent. The block-shapedPLGA/TCP composite material was pulverized in a pulverizer and filtratedwith a sieve of 40 to 60 meshes to obtain a polymer particle with adiameter ranging from 250 to 440 μm. The PLGA/TCP composite particleswere dry mixed with NaCl particles in a weight ratio of 20/80%.

The above round slice of NaCl particles-contained PLGA was placed in around-shaped teflon filtering flask having a diameter of 8.5 mm with thelower end thereof connected to an exhaust device. The PLGA strip wassurrounded the periphery of the round slice in the Teflon filteringflask to form a round fillister. 0.05 g of NaCl particles were filledinto the fillister and tightly flattened. 0.5 g of the mixture ofPLGA/TCP composite particles and NaCl particles were then filled therest space and tightly flattened. The Teflon mold was poured withacetone to immerse the mixture of particles. A negative pressure wasexerted by turning on the exhaust valve to draw out superfluous solvent.This step enables the partially dissolved polymer particles to adhere toone another. When an integral structure was formed, a large amount ofdeionized water was poured onto the top of the filter and the exhaustvalve was turned on again to pass the large amount of water through thematerials to dialyze and solidify the polymer particles. At the sametime, NaCl particles in the interior were washed out by water. Thesolidified matrix was obtained from the filter and immersed and stirredin a large beaker containing deionized water in which the deionizedwater was changed every six hours under room temperature for one day towash out the remaining solvent and salt particles. The solidified matrixwas then heat dried in a vacuum oven at 50° C. for one day and amulti-layered porous matrix having an upper hollow cavity was obtained.The multi-layered porous matrix was immersed in 75% alcohol for 6 hoursand then in a substantial amount of sterilized phosphate buffered saline(PBS) to replace the alcohol.

The prepared multi-layered porous matrix is shown in FIGS. 1A and 2B.FIG. 1A is a lateral view and FIG. 1B is a cross section of the preparedmulti-layered porous matrix. FIGS. 1A and 1B show a two-layeredstructure with an upper hollow cavity surrounded by a thick wall and alower evenly porous structure. The multi-layered porous matrix was alsoobserved under electron microscope and the results were shown in FIG.2A˜2C. FIG. 2A shows the porous structure surrounding the hollow cavitywith the pore size and the porosity of 112±41 μm and 84.2±2.4 vol %respectively, FIG. 2B shows the porous structure beneath the hollowcavity with the pore size and the porosity of 115±57 μm and 86.6±3.1 vol% respectively, and FIG. 2C shows the interface between the porousstructures surrounding and beneath the hollow cavity. The porousstructure of the interface is interconnected and no closed interface wasobserved.

Example 2 Isolation of Cartilage Tissue and Enzyme Treatment

Eight Lee-Sung strain adult (about 12-month-old) miniature pigs withweight of 50˜65 kg each were used (half male/half female). The pigs wereanesthetized with Pentothal (powder, 2.5 g/ampoule, Abbott, diluted withnormal Saline to 100 m1/ampoule) by I.V. injection (marginal ear vein).The surgical site was shaved and sterilized with alcoholic povidineiodine. A vertical incision was made on the midline of the knee jointand extends inside to expose the knee joint. Autologous cartilage wascollected with a curette from the unstressed site of the joint until thesubchondral bone was exposed. The collected autologous cartilage wasplaced in a 10-cm petri dish and minced with a scalpel. The mincedcartilage tissues were filtrated with a sieve of 20-40 meshes to be in asize ranging 560-800 μm, as shown in FIG. 3A. The filtrated cartilagetissues were placed in a 15-m1 centrifugation tube and treated with 5 mlof collagenase (2 mg/m1 PBS) at 37° C. for 4 hours to partially releasethe chondrocytes, as shown in FIG. 3B. The dissolved cartilage tissueswere centrifuged at 1500 r.p.m. for 5 minutes to separate thechondrocytes and collagenase. Supernatant containing collagenase wasdiscarded and the remaining cartilage tissues were washed with PBS twiceand centrifuged twice to totally remove the collagenase.

Example 3 In Vivo Implantation of the Multi-Layered Matrix Containingthe Cartilage Tissue

The enzyme treated cartilage tissues were injected into the hollowcavity of the multi-layered porous matrix by an 18-gauge syringe. Thetissue blocks may centralize in the upper cavity since they are largerthan the pore size of the matrix. A biphasic matrix with the uppercavity containing cartilage tissues and chondrocytes and the lowerporous matrix were formed.

3-1: Implantation of Nude Mice

To determine whether the implant containing the cartilage tissues formstwo layers of cartilage and bone in vivo, the biphasic matrix wasimplanted into the back of nude mice and the histological morphology wasobserved. The operation is as described below. The back of a nude mousewas locally anesthetized and sterilized. An incision with a size of0.5-1 cm was made subcutaneously, and the biphasic matrix containing thecartilage tissues was implanted into the subcutaneous site of the backof the nude mouse. The incision was then sutured. The nude mouse wasraised in its own cage. After 4 weeks, the implant was taken out fromthe nude mouse and analyzed. The matrix, as shown in FIGS. 4A and 4B, isintact and a new cartilage tissue is grown at the site of the hollowcavity. The histological results are shown in FIG. 5A˜5E. FIGS. 5A and5B are HE staining and the results reveal a newly formed cartilagetissue layer at the site of the hollow cavity extended toward the lowerporous structure to form an interface between cartilage and bone. Undera magnified field, as shown in FIG. 5B, the implanted cartilage tissueswere apparently fused and the tissue blocks did not have a clearinterface. FIGS. 5C and 5D are safranin-O and type II collagen stainingrespectively, and FIG. 5E is type I collagen staining. The results showapparent safranin-O and type II collagen stain with no type I collagenstain, indicating that the cartilage tissue at the hollow cavity secretshyaline cartilage specific substance such as type II collagen and doesnot dedifferentiate into fibrocartilages.

3-2: Implantation of Pig

The mid-portion of the distal articular surface of the lateral andmedial condylus of femur of a Lee-Sung strain miniature pig was drilledto form a cylindrical hole with a diameter and depth of 8.5 mm. Thecartilage and bone tissues including the whole layer of cartilage tissuewith a thickness of 2 mm, the subchondral bone, and partial sponge bonein the cylindrical pore were removed, as shown in FIG. 6A. The controlgroup was implanted with a matrix with no tissue, and the experimentalgroup was implanted with the above biphasic matrix with theenzyme-treated autologous cartilage tissues. The implant should fit thecylindrical hole and would not drop out from the hole due to thepress-fitting. The articular surface and the implant surface should beat the same level, to avoid step-off, as shown in FIG. 6B. The joint waslavaged and sutured layer by layer. After the surgery, the pig wasallowed to stand with four legs. Six months later, the knee joints ofthe control and experimental groups were observed and the results, asshown in FIG. 7, reveal that the control group (left side) has someregeneration around the defect but could not be completely repaired. Theright side is the experimental group and it shows that the defect siteis covered with a layer of new cartilage tissues. The joints wereobserved under X-ray photography as shown in FIGS. 8A and 8B. FIG. 8Ashows that the control group has no bone tissue extended into theimplant, and FIG. 8B shows that the experimental group has apparent newbone tissue extended into the implant.

By the results given above, the biphasic implant prepared by theembodiment of the method of the invention effectively formed a newcartilage layer at the hollow cavity of the matrix containing cartilagetissues. From the implantation of the animal model of full thicknessdefect, it shows that the implant of the embodiment of the inventioneffectively repair full thickness defects.

While the invention has been described by way of example and in terms ofthe preferred embodiments, it is to be understood that the invention isnot limited to the disclosed embodiments. To the contrary, it isintended to cover various modifications and similar arrangements (aswould be apparent to those skilled in the art). Therefore, the scope ofthe appended claims should be accorded the broadest interpretation so asto encompass all such modifications and similar arrangements.

1. A method of repairing tissue comprising the steps of: i) providing amulti-layered matrix comprising a first element and a second elementconnected thereto comprising a hollow cavity, wherein the first and thesecond elements are composed of a composite material comprising abioabsorbable porous material; ii) seeding a cell block into the hollowcavity of the second element of the multi-layered matrix, wherein thecell block is enzyme-treated for 4 hours before being seeded into thehollow cavity; and iii) directly implanting the multi-layered matrixcontaining the cell block into a defect site of a living subject withoutan ex vivo incubation of the multi-layered matrix containing the cellblock.
 2. The method as claimed in claim 1, wherein the bioabsorbableporous material comprises polylactic acid (PLA), polyglycolic acid(PGA), poly (glycolide co- lactide) (PLGA), polyanhydride,polycaprolactone (PCL), polydiester, polyorthoester, collagen, gelatin,hyaluronic acid, chitosan, or polyethylene glycol (PEG), or a mixturethereof.
 3. The method as claimed in claim 1, wherein the compositematerial further comprises a second material, wherein the secondmaterial comprises hydroxyapatite (HAP), tricalcium phosphate (TCP),tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA),dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), orcalcium pyrophosphate (CPP), or a mixture thereof.
 4. The method asclaimed in claim 1, wherein the porous material has a pore size ranging50 to 1,000 μm.
 5. The method as claimed in claim 1, wherein the cellblock is a minced tissue block.
 6. The method as claimed in claim 1,wherein the cell block is a tissue block which is minced andenzyme-treated to release partial cells.
 7. The method as claimed inclaim 1, wherein the enzyme is collagenase, hyaluronidase, trypsin, orproteinase, or a mixture thereof.
 8. The method as claimed in claim 1,wherein the defect site is a joint.
 9. The method as claimed in claim 8,wherein the cell block is a cartilage tissue.
 10. The method as claimedin claim 9, wherein the enzyme is collagenase.
 11. The method as claimedin claim 1, wherein the cell block is a cell aggregate.
 12. The methodas claimed in claim 1, wherein the cell block is a granular carrierattached with a cell.
 13. The method as claimed in claim 1, wherein thesize of the cell block is larger than the pore size of the porousmaterial.
 14. The method as claimed in claim 1, wherein the cell blockhas a size ranging 100 to 2,000 μm.